Genome Reconstruction: A Puzzle with a Billion Pieces

1. Genome Reconstruction: A Puzzle with a Billion Pieces Phillip Compeau & Pavel Pevzner University of California-San Diego

2. Outline • Introduction to Genome Sequencing • The Newspaper Problem • DNA Chips: A First Shot at Sequencing with Short Reads • Two Mathematical Detours • Introduction to Graph Theory • Euler’s Theorem • ECP vs. HCP and Algorithmic Complexity • From Euler and Hamilton to Fragment Assembly • De Bruijn and a Final Solution to Fragment Assembly • Generalizing Fragment Assembly

3. Section 1: Introduction to Genome Sequencing

4. What Is Genome Sequencing? • A genome can be represented as a book written in an alphabet containing only 4 letters, called nucleotides: A,T,G, and C. • A human genome has roughly 3 billion nucleotides. • Genome sequencing is the process of determining the sequence of nucleotides that make up a genome. ...CTGATGATGGACTACGCTACTACTGCTAGCTGTATTACGATCAGCTACCACATCGTAGCTACGATGCATTAGCAAGCTATCGATCGATCGATCGATTATCTACGATCGATCGATCGATCACTATACGAGCTACTACGTACGTACGATCGCGGGACTATTATCGACTACAGATAAAACATGCTAGTACAACAGTATACATAGCTGCGGGATACGATTAGCTAATAGCTGACGATATATAGCCGAGCGGCTACGATGATGCTAGCTGTACAGCTGATGATCTAGCTATCGATGCGATCGATGCGCGAGTGCGATCGATCACTTCGAGCTAGCTGATCGATCGATGCTAGCTAGCTGACTGATCATGGCGTTAGCTAGCTAGCTGATCGTCGATCGTACGTAGCTGATTACGATCGTCCGATCGTGCTATGACGTACGAGGCGGCTACGTAGCATGCTAGCTGACTGATGTAGCTAGCTATACGATACTATATATTCGATCGATTTATTACCATGACTGACGCGCATCGCTGTACACGTACTAGCTGATCGATGCTAGTCGATCGATCGATCATGTTATATATCGCGGCGCATCGATCGACTGCTCGATTATCGATACGTCGATCGCTGTATATACGTCTTTATAGCTAGGAGCATAGCGACGCGCTATCGATCGATCGTCTAGTCGACTGATCGTACTAGCTGACGCTGACGACTAGCTAGCTATCGACGATCGTAGTGCGATTACTAGCTAGGATCCTACTGTACGTCAGTCAGTCTGATCGATAGCGAGGAAAGCGAGACTGATCGTTCTCTAGATGTAGCTGATGTGACTACTATACTACTGGCAGCGATCGGGA…

5. What Is Genome Sequencing? • Different people have slightly different genomes: all humans share 99.9% of the same genetic code. • The 0.1% difference accounts for height, eye color, high cholesterol susceptibility, etc. CTGATGATGGACTACGCTACTACTGCTAGCTGTATTACGATCAGCTACCACATCGTAGCTACGATGCATTAGCAAGCTATCGATCGATCGATCGATTATCTACGATCGATCGATCGATCACTATACGAGCTACTACGTACGTACGATCGCGGGACTATTATCGACTACAGATAAAACATGCTAGTACAACAGTATACATAGCTGCGGGATACGATTAGCTAATAGCTGACGATATCCGAT CTGATGATGGACTACGCTACTACTGCTAGCTGTATTACGATCAGCTACAACATCGTAGCTACGATGCATTAGCAAGCTATCGATCGATCGATCGATTATCTACGATCGATCGATCGATCACTATACGAGCTACTACGTACGTACGATCGCGTGACTATTATCGACTACAGATGAAACATGCTAGTACAACAGTATACATAGCTGCGGGATACGATTAGCTAATAGCTGACGATATCCGAT

6. Species Sequencing vs. Individual Genome Sequencing • Species Sequencing: Determine the “consensus genome” of an entire species.

7. Species Sequencing vs. Individual Genome Sequencing • Individual Sequencing: Determine how an individual differs from its species.

8. Why Would We Want to Sequence a Genome? • Species genome sequencing: • Compare various species (e.g. human and chimpanzee) to understand how their genes function (e.g. which genes are important for braindevelopment). • Reveal evolutionaryrelationships betweenspecies. • Determine the geneticmakeup of ourevolutionary ancestors.

9. Why Would We Want to Sequence a Genome? • Individual genome sequencing: • Unearth the genetic basis of many diseases. • Forensics applications. • Example: In 2010, 6-year old Nicholas Volker became the first human being to be saved because of genome sequencing. • Doctors could not diagnose his condition, which caused strange infections; he went through nearly 100 surgeries. • Genome sequencing revealed a rare mutation in a gene linked to a defect inhis immune system. • This led doctors to use advancedimmunotherapy, which saved the child.

10. Brief History of Genome Sequencing • Late 1970s: Walter Gilbert and Frederick Sanger develop independent sequencing methods. • 1980: They share the Nobel Prize in Chemistry. • Still, their sequencing methods were too expensive for large genomes: with a $1 per nucleotide cost, it would cost $3 billion to sequence the human genome. Walter Gilbert Frederick Sanger

11. Brief History of Genome Sequencing • 1990: The public Human Genome Project, headed by Francis Collins, aims to sequence the human genome. • 1997: Craig Venter founds Celera Genomics, a private firm, with the same goal. Francis Collins Craig Venter

12. Brief History of Mammalian Genome Sequencing • 2000: The draft of the human genome is simultaneously completed by the (public) Human Genome Consortium and (private) Celera Genomics.

13. Brief History of Mammalian Genome Sequencing • 2000s: Many more mammalian genomes are sequenced.

14. The Arrival of Personal Genomics • 2000s: Many companies launch projects aimed at reducing sequencing costs by orders of magnitude. • 2010: The market for sequencing machines takes off. • Illumina reduces the cost of sequencing an individual human genome from $3 billion to $10,000. • Complete Genomics builds a genomic factory in Silicon Valley that sequences hundreds of genomes per month. • Beijing Genome Institute orders hundreds of sequencing machines, becoming the world’s largest sequencing center. • 23andMe offers partial genome sequencing for $499. • Many universities introduce new courses in which students study their own genomes.

15. The Future of Genome Sequencing • 2010s?: Genome sequencing will hopefully continue to bloom. • The $1,000 human genome may arrive as early as in 2012. • Hopefully, sequencing an individual genome will soon become as routine as an X-ray.

16. What Makes Genome Sequencing So Difficult? • When we read a book, we can read the entire book one letter at a time from the beginning to the end. • However, modern sequencing machines cannot read an entire genome one nucleotide at a time from beginning to end. They can only shred the genome and read the short pieces. • Thus, we can identify very short fragments of DNA (~100 nucleotides long), called reads. • But we have no idea which genomic positions these reads come from! • We must figure out how to put the reads back together to assemble a genome.

17. Section 2: The Newspaper Problem and Genome Sequencing

18. The Newspaper Problem

19. The Newspaper Problem

20. The Newspaper Problem

21. The Newspaper Problem

22. The Newspaper Problem

23. The Newspaper Problem

24. The Newspaper Problem as an “Overlap Puzzle” • The newspaper problem is not the same as a jigsaw puzzle: • We have multiple copies of the same edition of a newspaper. • Plus, some pieces of paper got blown to bits in the explosion. • Instead, we must use overlapping shreds of paper to reconstruct what the newspaper said. • This gives us a giant overlap puzzle!

25. Sequencing is Harder than Newspaper Problem • In the newspaper problem, we have the rules of language and common sense (e.g. “murder” and “suspect” would often appear near each other in a newspaper.) • However, the “language” of DNA remains largely unknown.

26. Sequencing is Harder than Newspaper Problem • There are lots of repeated substrings in every genome (50% of human genome is formed by repeats). • Example: GCTTis repeated 4 times in the following: • AAGCTTCTATTGCTTAATTGGCTTGCTTCGCTTTG • Analogy: The Triazzle puzzle contains lots of repeated figures. This makes it very difficult tosolve (even with just 16 pieces).

27. Sequencing a Genome: Lab + Computation • Read Generation (Experimental):Generate many reads from multiplecopies of the same genome. • Fragment Assembly (Computational):Use these reads to algorithmicallyput the genome back together.

28. Sequencing a Genome: Illustration Multiple (Unsequenced) Genome Copies

29. Sequencing a Genome: Illustration Multiple (Unsequenced) Genome Copies Read Generation

30. Sequencing a Genome: Illustration Multiple (Unsequenced) Genome Copies Read Generation Reads

31. Sequencing a Genome: Illustration Multiple (Unsequenced) Genome Copies Read Generation Reads Fragment Assembly

32. Sequencing a Genome: Illustration Multiple (Unsequenced) Genome Copies Read Generation Reads Fragment Assembly Sequenced Genome …GGCATGCGTCAGAAACTATCATAGCTAGATCGTACGTAGCC…

33. Section 3: DNA Chips: A First Shot at Sequencing with Short Reads

34. DNA Chips: From an Idea to a New Industry • 1989:RadojeDrmanac, AndreyMirzabekov, and Edwin Southern independently invent DNA chips (arrays) for read generation. • Key Idea: Generate all k-mers (see below) from the genome in the hope that they can be assembled to reconstruct the genome. • 1989: Science magazine writes, “Using DNA arrays for sequencing would simply be substituting one horrendous task for another.” • 2000: Arrays are a multi-billion dollar industry Mirzabekov Drmanac Southern k-mer: A string of length k (in an alphabet of 4 nucleotides)

35. DNA Chips: Implementation • Synthesizea distinct k-mer in each of 4k cells in the array. • Cover the array with multiple copies of a fluorescently-labeled unknown DNA fragment. • DNA will hybridize witha k-mer if it contains thecomplement of that k-mer. • Use a spectroscope todetermine which sites emitlight …the complementsof these sites will reveal thek-mers in the unknownDNA fragment = our reads!

36. DNA Chips: Illustration

37. DNA Chips: Example • What are our reads?

38. DNA Chips: Example • What are our reads? CAT

39. DNA Chips: Example • What are our reads? CAT ||| ATG

40. DNA Chips: Example • What are our reads? CAT ATG

41. DNA Chips: Example • What are our reads? CAT ATG

42. DNA Chips: Example • What are our reads? CAT ATG

43. DNA Chips: Example • What are our reads? CAT ATG

44. DNA Chips: Example • What are our reads? • So 3-mer ATG mustoccur in the genome! ATG

45. Red 3-mers Must Occur in the Genome • What are our reads? CAC  GTG CGC  GCG • CAT  ATG TGC  GCA ACG  CGT ATT  AAT CCA  TGG GCA  TGC GCC  GGC TTG  CAA

46. Red 3-mers Must Occur in the Genome • What are our reads? • CAC CGC  GCG • CAT  ATG

47. Red 3-mers Must Occur in the Genome • What are our reads? • CAC  GTG CGC  GCG • CAT  ATG

48. Red 3-mers Must Occur in the Genome • What are our reads? • CAC  GTG • CGC • CAT  ATG • TGC  GCA • ACG  CGT • ATT  AAT • CCA  TGG • GCA  TGC • GCC  GGC • TTG  CAA

49. Red 3-mers Must Occur in the Genome • What are our reads? • CAC  GTG • CGC  GCG • CAT  ATG • TGC  GCA • ACG  CGT • ATT  AAT • CCA  TGG • GCA  TGC • GCC  GGC • TTG  CAA

50. Red 3-mers Must Occur in the Genome • What are our reads? • CAC  GTG • CGC  GCG • CAT  ATG • TGC